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Human Invasive Muscular Sarcocystosis Induces Th2 Cytokine

Polarization and Biphasic Cytokine Changes, Based on an

Investigation among Travelers Returning from Tioman Island,
Dennis Tappe,a Günther Slesak,b José Vicente Pérez-Girón,c Johannes Schäfer,b Andreas Langeheinecke,d Gudrun Just-Nübling,e
César Muñoz-Fontela,a,c,f Kerstin Püllmanng
Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germanya; Tropenklinik Paul-Lechler-Hospital, Department of Tropical Medicine, Tübingen, Germanyb; Heinrich
Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germanyc; Medizinische Klinik 1, Klinikum Saarbrücken, Saarbrücken, Germanyd; Klinik II, Centre for
Infectious and Tropical Diseases, J.W. Goethe University, Frankfurt/Main, Germanye; German Centre for Infection Research (DZIF), partner site Hamburg-Luebeck-Borstel,
Hamburg, Germanyf; AescuLabor Hamburg, Hamburg, Germanyg

Sarcocystis nesbitti is a parasite responsible for a biphasic eosinophilic febrile myositis syndrome in two recent outbreaks in Ma-
laysia. We demonstrate Th2 cytokine polarization in infected travelers, an overall cytokine production decrease in the early
phase of the disease suggestive of initial immunosuppression, and elevated levels of proinflammatory and chemotactic cytokines
in the later myositic phase.

M uscular sarcocystosis, a parasitic zoonosis that has only

rarely been reported clinically in humans in Southeast Asia
(1), has come to recent attention because of two concomitant
elers were diagnosed with phase one of the illness (2 male, 1 fe-
male; 15 to 41 years old), and 6 travelers were diagnosed with
phase two of the disease (1 male, 5 female; 9 to 44 years old). In all
outbreaks in Malaysia. One outbreak occurred in 2012 on Pang- patients, multiplex cytokine serum analyses (Bio-Rad Laborato-
kor Island, located on the west coast of peninsular Malaysia (2, 3), ries, Munich, Germany) were performed, and 10 sera from
and involved 89 students and teachers, mostly from Asia. The healthy blood donors were run in parallel. Serum cytokine level
other one took place in two successive waves in 2011 and 2012 on measurements from all 9 patients demonstrated no differences
Tioman Island, located on the east coast, and affected ⬎100 inter- between phases one and two and the sera from healthy blood
national travelers (4–7). A third wave of infected travelers return- donors for interleukin 4 (IL-4), IL-10, tumor necrosis factor alpha
ing from Tioman Island was reported in 2014 (8), indicating an (TNF-␣), and gamma interferon (IFN-␥) (data not shown). How-
ongoing transmission from a still unknown source. Transmission ever, sera of patients in phase one exhibited lower concentrations
on Pangkor Island has been linked to contaminated freshwater of IL-2, IL-7, IL-12, IL-15, and IL-17 than the healthy controls,
(3). Both outbreaks have been caused by Sarcocystis nesbitti (2, 7), with no striking difference in phase two sera (Fig. 1). Also, sera
a possibly snake-associated, intracellular protozoan parasite (9, from phase one had lower concentrations of RANTES, monocyte
10). In the natural cycle of Sarcocystis sp., intramuscular encysta- chemoattractant protein 1 (MCP-1), platelet-derived growth fac-
tion of the parasite occurs in the intermediate host after oral in- tor (PDGF), and vascular endothelial growth factor (VEGF) than
gestion of infective oocysts which are fecally excreted by the final sera from phase two and healthy controls (Fig. 1). Sera from phase
host. The disease has a two-phase symptomatology (3, 6, 7) con- two, in contrast, showed elevations of IL-5, IL-6, IL-8, IL-9, and
sisting of an early phase characterized by fever, headache, and MIP-1␣ levels, compared to those in sera from phase one and
myalgia (phase one), followed by an asymptomatic or less symp- healthy controls. MIP-1␤ levels, in contrast, were lower in phase
tomatic interval, and a later, again symptomatic myositic phase one sera than in the healthy controls. In sera from both phases one
(phase two). Severe symptoms and prolonged and relapsing and two, IP-10 was also elevated and, by trend, IL-1␤ was also (Fig.
courses, even over months, have been documented (3, 6, 7, 11). 2). Of note, two age-, sex-, and family-unrelated patients from the
Unusual for a protozoal parasite, the organism causes eosinophilia
in the human accidental intermediate host. Here, we analyze lym-
phocyte and cytokine responses in infected German travelers re- Received 19 January 2015 Returned for modification 2 March 2015
Accepted 16 April 2015
turning from Tioman Island in different clinical phases of the
Accepted manuscript posted online 22 April 2015
Citation Tappe D, Slesak G, Pérez-Girón JV, Schäfer J, Langeheinecke A, Just-
In May and June 2014, 9 patients were seen in different travel Nübling G, Muñoz-Fontela C, Püllmann K. 2015. Human invasive muscular
clinics in Germany with a febrile myositis syndrome after travel to sarcocystosis induces Th2 cytokine polarization and biphasic cytokine changes,
Tioman Island (2°48=47⬙N, 104°11=17⬙E). The clinical picture was based on an investigation among travelers returning from Tioman Island,
identical to that of invasive muscular sarcocystosis previously re- Malaysia. Clin Vaccine Immunol 22:674 –677. doi:10.1128/CVI.00042-15.
ported from the island. Creatine kinase and eosinophil counts Editor: P. P. Wilkins
were elevated in all patients, and trichinellosis serology was nega- Address correspondence to Dennis Tappe,
tive. All patients had stayed in various villages at the northern tip Copyright © 2015, American Society for Microbiology. All Rights Reserved.
of the island, as reported previously (6). According to the clinical doi:10.1128/CVI.00042-15
course and the two-phase nature of invasive sarcocystosis, 3 trav-

674 Clinical and Vaccine Immunology June 2015 Volume 22 Number 6
Cytokine Changes in Human Muscular Sarcocystosis

FIG 1 Cytokine levels in travelers with muscular sarcocystosis compared to healthy controls. Box and whisker plots showing median, upper, and lower quartile,
minimum, and maximum values. Sera of patients in phase one (early phase) had a tendency to exhibit lower concentrations of the cytokines depicted than healthy
controls and sera from phase two (late phase). IL-2 and IL-15 levels in phase one sera cluster at the bottom line. IL, interleukin; MCP-1, monocyte chemotactic
protein 1; VEGF, vascular endothelial growth factor; PDGF, platelet-derived growth factor; RANTES, regulated on activation, normal T cell expressed and

phase two cohort exhibited the maximum levels of most of the CD8⫹ ratio) but low B cell counts (5.3% [109 cells/␮l] and 6.2%
cytokines measured. When the data of these two patients were [65 cells/␮l]). Lymphocyte cytokine production and accumula-
substracted, the cytokine elevations became less pronounced than tion were also assessed by flow cytometry. For intracellular cyto-
those of phase one sera (not shown). Flow cytometry lymphocyte kine staining (13), stimulation with phorbol-myristate-13-acetate
phenotyping (Navios; Beckman Coulter, Krefeld, Germany) by (PMA) and ionomycin was performed, followed by staining with
following standard procedures (12) was performed in 2 patients in monoclonal antibodies against CD3 ECD (energy coupled dye,
the late second phase of the above-described cohort (1 male, 1 phycoerythrin [PE] and Texas Red), CD8 PC7, CD69 ECD, IL-2
female; 27 and 28 years old). Both patients showed normal relative PE, IFN-␥ (Beckman Coulter), IL-17A fluorescein isothiocyanate
counts of natural killer (NK) cells, total T lymphocytes, activated (FITC), and IL-4 PE (BioLegend, Fell, Germany). In-house age-
T lymphocytes, and CD4⫹ and CD8⫹ T cells (normal CD4⫹/ and sex-adapted reference values from healthy blood donors were

June 2015 Volume 22 Number 6 Clinical and Vaccine Immunology 675
Tappe et al.

FIG 2 Cytokine levels in travelers with muscular sarcocystosis compared to healthy controls. Box and whisker plots showing median, upper, and lower quartile,
minimum, and maximum values. Sera of patients in phase two (later phase) had a tendency to have higher levels of the cytokines depicted than healthy controls
and, except for IP-10, sera from phase one (early phase). IL, interleukin; IP-10, interferon gamma-induced protein 10; MIP-1␣/␤, macrophage inflammatory
protein 1 alpha/beta.

established. Both patients showed a decrease of IL-2 and IFN-␥ crease in T cell function and a decrease in dendritic cell activation
production by CD4⫹ lymphocytes and an age- and sex-related and recruitment (14) in this initial phase of the infection. A similar
normal IL-4 expression. Both patients had eosinophilia (11% each T cell immunosuppression is seen in acute infections with the
[580 cells/␮l]) and basophilia (1.9% [110 cells/␮l] and 4.5% [240 related parasite Toxoplasma gondii in a mouse model (15). The
cells/␮l]) at the time of the analysis. respective cytokine levels returned to values comparable with
We here demonstrate in the two-phase disease of human inva- those of the healthy blood donor cohort in phase two of the dis-
sive sarcocystosis a decrease of several serum cytokines in phase ease. In contrast to phase one, in phase two of the infection several
one, including the Th1 cytokines IL-2 and IL-12, paralleled by proinflammatory and chemotactic cytokines are elevated, to-
normal levels of IFN-␥. Low levels of RANTES, MCP-1, and IL- gether with IL-5, a Th2 cytokine and inductor of eosinophilia. The
12p70 and further regulatory cytokines may point toward a de- biphasic changes in cytokine levels observed during the two clin-

676 Clinical and Vaccine Immunology June 2015 Volume 22 Number 6
Cytokine Changes in Human Muscular Sarcocystosis

ical phases of invasive sarcocystosis likely reflect the hosts’ im- 5. Esposito DH, Freedman DO, Neumayr A, Parola P. 2012. Ongoing
mune response during parasite invasion and formation of schi- outbreak of an acute muscular Sarcocystis-like illness among travellers
returning from Tioman Island, Malaysia, 2011-2012. Euro Surveill 17:
zonts in the vascular endothelium (assumed phase one of the pii⫽20310.
clinical symptomatology), followed by the development of sarco- ⫽20310.
cysts in muscles (phase two). Eosinophilia, a hallmark of human 6. Slesak G, Tappe D, Keller C, Cramer J, Güthoff W, Zanger P, Frank M,
muscular sarcocystosis (1–8), is unusual in unicellular invasive Ernestus K, Rauthe S, Stich A, Schäfer J. 2014. Muscular sarcocystosis
parasitic infections and is rather seen in tissue-invasive infections after travel to Malaysia: a case series from Germany. Dtsch Med Wochen-
schr 139:990 –995.
involving multicellular parasites, allergy, and hematological dis- 7. Esposito DH, Stich A, Epelboin L, Malvy D, Han PV, Bottieau E, da
orders. Levels of IL-4 and IL-10, additional Th2 cytokines, were Silva A, Zanger P, Slesak G, van Genderen PJ, Rosenthal BM, Cramer
not elevated, but the observed decrease of intracellular IL-2 and JP, Visser LG, Muñoz J, Drew CP, Goldsmith CS, Steiner F, Wagner N,
IFN-␥ following lymphocyte stimulation also points toward a Grobusch MP, Plier DA, Tappe D, Sotir MJ, Brown C, Brunette GW,
Fayer R, von Sonnenburg F, Neumayr A, Kozarsky PE, Tioman Island
slightly Th2-biased immune response. Characteristic biphasic im-
Sarcocystosis Investigation Team. 2014. Acute muscular sarcocystosis:
mune responses have also been described in eosinophilic muscle- an international investigation among ill travelers returning from Tioman
invasive helminth infections (trichinellosis), with a Th1 response Island, Malaysia, 2011-2012. Clin Infect Dis 59:1401–1410. http://dx.doi
during the initial intestinal phase and a later Th2 response during .org/10.1093/cid/ciu622.
the muscle stage (16). Interestingly, B cell counts in the two exam- 8. Tappe D, Stich A, Langeheinecke A, von Sonnenburg F, Muntau B, Schäfer J,
Slesak G. 2014. Suspected new wave of muscular sarcocystosis in travellers return-
ined patients were low. ing from Tioman Island, Malaysia, May 2014. Euro Surveill 19:pii⫽20816. http:
This study has several limitations. First, the number of patients //⫽20816.
examined was small, thus diminishing the strength of the obser- 9. Tian M, Chen Y, Wu L, Rosenthal BM, Liu X, He Y, Dunams DB, Cui
vation. Second, the lymphocyte analyses were performed in phase L, Yang Z. 2012. Phylogenetic analysis of Sarcocystis nesbitti (Coccidia:
two patients only, and no data on patients in an earlier phase of Sarcocystidae) suggests a snake as its probable definitive host. Vet Parasitol
sarcocystosis were obtained. Despite the fact that we show rather 10. Lau YL, Chang PY, Tan CT, Fong MY, Mahmud R, Wong KT. 2014.
preliminary data, to our knowledge this is the first study investi- Sarcocystis nesbitti infection in human skeletal muscle: possible transmis-
gating immune parameters in patients with sarcocystosis. Future sion from snakes. Am J Trop Med Hyg 90:361–364.
studies will have to address lymphocyte and cytokine responses in .4269/ajtmh.12-0678.
11. Slesak G, Schäfer J, Langeheinecke A, Tappe D. 2015. Prolonged clinical
more detail, including stimulation of immune cells with Sarcocys- course of muscular sarcocystosis and effectiveness of cotrimoxazole
tis antigen. among travelers to Tioman Island, Malaysia, 2011-2014. Clin Infect Dis
REFERENCES 12. Calvelli T, Denny TN, Paxton H, Gelman R, Kagan J. 1993. Guideline
1. Arness MK, Brown JD, Dubey JP, Neafie RC, Granstrom DE. 1999. An for flow cytometric immunophenotyping: a report from the National In-
outbreak of acute eosinophilic myositis attributed to human Sarcocystis stitute of Allergy and Infectious Diseases, Division of AIDS. Cytometry
parasitism. Am J Trop Med Hyg 61:548 –553. 14:702–715.
2. Abubakar S, Teoh BT, Sam SS, Chang LY, Johari J, Hooi PS, Lakhbeer-Singh 13. Mascher B, Schlenke P, Seyfarth M. 1999. Expression and kinetics of cyto-
HK, Italiano CM, Omar SF, Wong KT, Ramli N, Tan CT. 2013. Outbreak of kines determined by intracellular staining using flow cytometry. J Immunol
human infection with Sarcocystis nesbitti, Malaysia, 2012. Emerg Infect Dis Methods 223:115–121.
19:1989 –1991. 14. Sallusto F, Lanzavecchia A, Mackay CR. 1998. Chemokines and chemokine
3. Italiano CM, Wong KT, AbuBakar S, Lau YL, Ramli N, Syed Omar SF, receptors in T-cell priming and Th1/Th2-mediated responses. Immunol Today
Kahar Bador M, Tan CT. 2014. Sarcocystis nesbitti causes acute, relapsing 19:568–574.
febrile myositis with a high attack rate: description of a large outbreak of 15. Tenorio EP, Fernández J, Castellanos C, Olguín JE, Saavedra R. 2011. CD4⫹
muscular sarcocystosis in Pangkor Island, Malaysia, 2012. PLoS Negl Trop Foxp3⫹ regulatory T cells mediate Toxoplasma gondii-induced T-cell sup-
Dis 8:e2876. pression through an IL-2-related mechanism but independently of IL-10.
4. Tappe D, Ernestus K, Rauthe S, Schoen C, Frosch M, Müller A, Stich Eur J Immunol 41:3529 –3541.
A. 2013. Initial patient cluster and first positive biopsy findings in an 16. Yu YR, Deng MJ, Lu WW, Jia MZ, Wu W, Qi YF. 2013. Systemic
outbreak of acute muscular Sarcocystis-like infection in travelers returning cytokine profiles and splenic toll-like receptor expression during
from Tioman island, Peninsular Malaysia, in 2011. J Clin Microbiol 51: Trichinella spiralis infection. Exp Parasitol 134:92–101.
725–726. /10.1016/j.exppara.2013.02.014.

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